ABSTRACT
In pancreatic beta-cells, voltage-dependent K(+) (Kv) channels are potential mediators of repolarization, closure of Ca(2+) channels, and limitation of insulin secretion. The specific Kv channels expressed in beta-cells and their contribution to the delayed rectifier current and regulation of insulin secretion in these cells are unclear. High-level protein expression and mRNA transcripts for Kv1.4, 1.6, and 2.1 were detected in rat islets and insulinoma cells. Inhibition of these channels with tetraethylammonium decreased I(DR) by approximately 85% and enhanced glucose-stimulated insulin secretion by 2- to 4-fold. Adenovirus-mediated expression of a C-terminal truncated Kv2.1 subunit, specifically eliminating Kv2 family currents, reduced delayed rectifier currents in these cells by 60-70% and enhanced glucose-stimulated insulin secretion from rat islets by 60%. Expression of a C-terminal truncated Kv1.4 subunit, abolishing Kv1 channel family currents, reduced delayed rectifier currents by approximately 25% and enhanced glucose-stimulated insulin secretion from rat islets by 40%. This study establishes that Kv2 and 1 channel homologs mediate the majority of repolarizing delayed rectifier current in rat beta-cells and that antagonism of Kv2.1 may prove to be a novel glucose-dependent therapeutic treatment for type 2 diabetes.
Subject(s)
Insulin/metabolism , Islets of Langerhans/physiology , Potassium Channels, Voltage-Gated , Potassium Channels/physiology , 1-Methyl-3-isobutylxanthine/pharmacology , Adenoviridae/genetics , Animals , Cell Line , Cyclic AMP-Dependent Protein Kinases/metabolism , Delayed Rectifier Potassium Channels , Electric Conductivity , Gene Deletion , Gene Expression , Genetic Vectors , Glucose/pharmacology , Glyburide/pharmacology , Green Fluorescent Proteins , Insulin Secretion , Insulinoma/metabolism , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Kv1.4 Potassium Channel , Luminescent Proteins/genetics , Pancreatic Neoplasms/metabolism , Potassium Channel Blockers , Potassium Channels/genetics , RNA, Messenger/analysis , Rats , Recombinant Proteins , Shab Potassium Channels , Tetraethylammonium/pharmacology , TransfectionABSTRACT
In pancreatic beta-cells, glucose metabolism signals insulin secretion by altering the cellular array of messenger molecules. ATP is particularly important, given its role in regulating cation channel activity, exocytosis, and events dependent upon its hydrolysis. Uncoupling protein (UCP)-2 is proposed to catalyze a mitochondrial inner-membrane H(+) leak that bypasses ATP synthase, thereby reducing cellular ATP content. Previously, we showed that overexpression of UCP-2 suppressed glucose-stimulated insulin secretion (GSIS) in isolated islets (1). The aim of this study was to identify downstream consequences of UCP-2 overexpression and to determine whether insufficient insulin secretion in a diabetic model was correlated with increased endogenous UCP-2 expression. In isolated islets from normal rats, the degree to which GSIS was suppressed was inversely correlated with the amount of UCP-2 expression induced. Depolarizing the islets with KCl or inhibiting ATP-dependent K(+) (K(ATP)) channels with glybenclamide elicited similar insulin secretion in control and UCP-2-overexpressing islets. The glucose-stimulated mitochondrial membrane ((m)) hyperpolarization was reduced in beta-cells overexpressing UCP-2. ATP content of UCP-2-induced islets was reduced by 50%, and there was no change in the efflux of Rb(+) at high versus low glucose concentrations, suggesting that low ATP led to reduced glucose-induced depolarization, thereby causing reduced insulin secretion. Sprague-Dawley rats fed a diet with 40% fat for 3 weeks were glucose intolerant, and in vitro insulin secretion at high glucose was only increased 8.5-fold over basal, compared with 28-fold in control rats. Islet UCP-2 mRNA expression was increased twofold. These studies provide further strong evidence that UCP-2 is an important negative regulator of beta-cell insulin secretion and demonstrate that reduced (m) and increased activity of K(ATP) channels are mechanisms by which UCP-2-mediated effects are mediated. These studies also raise the possibility that a pathological upregulation of UCP-2 expression in the prediabetic state could contribute to the loss of glucose responsiveness observed in obesity-related type 2 diabetes in humans.
Subject(s)
Glucose/pharmacology , Insulin/metabolism , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Membrane Transport Proteins , Mitochondrial Proteins , Proteins/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/physiology , Animals , Dietary Fats/administration & dosage , Dietary Fats/pharmacology , Electrophysiology , Humans , Insulin Secretion , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Ion Channels , Mitochondria/drug effects , Mitochondria/physiology , Potassium Channels/metabolism , Proteins/genetics , RNA, Messenger/metabolism , Rats , Reference Values , Rubidium/metabolism , Uncoupling Protein 2ABSTRACT
Norcantharidin is the demethylated form of Cantharidin, which is the active ingredient of the blister beetle, Mylabris, a long used Chinese traditional medicine. Though not well publicized outside China, Norcantharidin is known to possess significant anti-hepatoma activity, and is relatively free from side effects. In the present study, glucose oxidation in tumour and liver tissue slices harvested from hepatoma-bearing animals was quantified by measuring the radioactivity of 14C-labelled CO2 released from 14C-glucose in oxygen-enriched incubation medium. Results were expressed as a tumour/liver ratio. For comparison, treatments with Norcantharidin, Adriamycin and with hepatic artery ligation were studied. The mean tumour/liver ratio was 4.2 +/- 2.2 in untreated controls, but dropped significantly to 2.3 +/- 0.5 (p < 0.05) with intra-arterial Norcantharidin (0.5 mg/kg) and to 2.3 +/- 0.7 (p < 0.05) with intra-arterial Adriamycin (2.4 mg/kg), and to 2.2 +/- 0.7 (p < 0.05) with hepatic artery ligation. However, with intravenous Adriamycin at 2.4 mg/kg, the mean tumour/liver ratio was reduced to only 3.5 +/- 2.0 and was not significantly different from untreated controls. It is concluded that intra-arterial Norcantharidin is as effective as intraarterial Adriamycin and hepatic artery ligation in suppressing tumour glucose oxidative metabolism. These result simply that Norcantharidin may have a role to play in the chemotherapy of primary liver cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Carbon Radioisotopes/metabolism , Carcinoma, Hepatocellular/drug therapy , Glucose/metabolism , Liver Neoplasms, Experimental/drug therapy , Animals , Antibiotics, Antineoplastic/therapeutic use , Carcinoma, Hepatocellular/metabolism , Doxorubicin/therapeutic use , Drug Evaluation, Preclinical , Hepatic Artery/surgery , Infusions, Intra-Arterial , Ligation , Liver Neoplasms, Experimental/metabolism , Male , Oxidation-Reduction , Rats , Rats, Inbred BUFABSTRACT
A prospective, comparative study was made between 371 patients undergoing laparoscopic cholecystectomy and 100 patients undergoing conventional cholecystectomy. Post-operative pain was assessed subjectively by a single observer using a visual analog score and objectively by assessment of parenteral analgesic used. Patients who underwent laparoscopic cholecystectomy required significantly less analgesia (46.7 mg vs 223.9mg mean pethidine dose, p < 0.01) and were observed to have mobilised earlier and had a shorter mean post-operative stay (3.5 days vs 5.9 days, p < 0.01). Laparoscopic cholecystectomy objectively reduces post-operative pain significantly and should be the new standard for treatment of gallstones.
